U.S. patent number 9,005,885 [Application Number 13/375,260] was granted by the patent office on 2015-04-14 for bioartificial lung.
This patent grant is currently assigned to The General Hospital Corporation. The grantee listed for this patent is Harald C. Ott. Invention is credited to Harald C. Ott.
United States Patent |
9,005,885 |
Ott |
April 14, 2015 |
**Please see images for:
( Certificate of Correction ) ** |
Bioartificial lung
Abstract
Presented is an airway organ bioreactor apparatus, and methods
of use thereof, as well as bioartificial airway organs produced
using the methods, and methods of treating subjects using the
bioartificial airway organs.
Inventors: |
Ott; Harald C. (Boston,
MA) |
Applicant: |
Name |
City |
State |
Country |
Type |
Ott; Harald C. |
Boston |
MA |
US |
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Assignee: |
The General Hospital
Corporation (Boston, MA)
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Family
ID: |
43298538 |
Appl.
No.: |
13/375,260 |
Filed: |
June 4, 2010 |
PCT
Filed: |
June 04, 2010 |
PCT No.: |
PCT/US2010/037379 |
371(c)(1),(2),(4) Date: |
February 24, 2012 |
PCT
Pub. No.: |
WO2010/141803 |
PCT
Pub. Date: |
December 09, 2010 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20120141439 A1 |
Jun 7, 2012 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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61184170 |
Jun 4, 2009 |
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61256281 |
Oct 29, 2009 |
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Current U.S.
Class: |
435/1.2; 435/373;
435/325; 435/284.1; 424/93.1; 435/1.1; 424/93.7 |
Current CPC
Class: |
A61L
27/3891 (20130101); C12M 25/14 (20130101); C12M
29/10 (20130101); A61L 27/3804 (20130101); C12M
41/30 (20130101); C12M 33/00 (20130101); C12M
33/04 (20130101); C12M 21/08 (20130101); A61M
16/0054 (20130101); A61M 2210/1039 (20130101) |
Current International
Class: |
A61K
35/42 (20060101); A01N 1/02 (20060101); A61K
35/12 (20060101); A61K 35/00 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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1555031 |
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Jul 2005 |
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EP |
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02/053193 |
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Jul 2002 |
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WO |
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2007/095192 |
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Aug 2007 |
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WO |
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2010/091188 |
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Aug 2010 |
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WO |
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Other References
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Primary Examiner: Lankford; Blaine
Assistant Examiner: Berke-Schlessel; David
Attorney, Agent or Firm: Fish & Richardson P.C.
Parent Case Text
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a 371 of International Application No.
PCT/US2010/037379, filed Jun. 4, 2010, and claims the benefit of
U.S. Provisional Application No. 61/184,170, filed on Jun. 4, 2009,
and U.S. Provisional Application No. 61/256,281, filed Oct. 29,
2009, the contents of which are incorporated by reference herein in
their entirety.
Claims
What is claimed is:
1. A method of providing a fuctional bioartificial airway organ,
the method comprising: providing a decellularized lung tissue
matrix comprising airway and vascular architecture; seeding a lumen
of the vascular architecture by perfusing endothelial cells through
the vascular architecture; seeding the lung tissue matrix with
epithelial cells over the airway architecture through a wet
ventilation line; providing the lung tissue matrix with wet
ventilation, through the airway architecture, and perfusion,
through the vascular architecture, for a time sufficient to produce
a wet-matured organ; and providing the wet-matured organ with dry
ventilation through the airway architecture, and with perfusion
through the vascular architecture, for a time sufficient for
providing a functional bioartifical lung that allows for the
exchange of oxygen.
2. The method of claim 1, wherein the epithelial cells are human
alveolar basal epithelial cells.
3. The method of claim 1, wherein the endothelial cells are
suspended in a fluid at a concentration of about 100 million cells
per 10 cc of fluid; and the epithelial cells are suspended in a
fluid at a concentration of about 100 million cells per 5 cc of
fluid.
4. The method of claim 1, further comprising monitoring the lung
tissue matrix until the time sufficient to produce a wet-matured
organ has passed; stopping the wet ventilation to the wet-matured
organ; applying an artificial surfactant to the wet-matured organ;
and starting the dry ventilation to the wet-matured organ.
5. The method of claim 1, wherein providing the lung tissue matrix
with wet ventilation comprises: connecting the airway architecture
to a wet ventilator via a wet ventilator line; connecting the lung
tissue matrix to a compliance chamber via a wet ventilation line;
increasing a wet airway pressure over the wet ventilation line; and
providing a wet positive and expiratory pressure (wPEEP) to the
lung tissue matrix by elevating the compliance chamber.
6. The method of claim 5, wherein the wet ventilation is provided
at a physiologic tidal volume.
7. The method of claim 1, wherein providing the wet-matured organ
with dry ventilation comprises: connecting the airway architecture
to a dry ventilation chamber via a dry ventilation line; connecting
the dry ventilation chamber to a first dry ventilator over a dry
positive and expiratory pressure (dPEEP) line; increasing a dry
airway pressure over the dry ventilation line; disconnecting the
wet ventilation line; and connecting the wet-matured organ to a
second dry ventilator via a dry ventilator line.
8. The method of claim 1, wherein the lung tissue matrix comprises
decellularized human lung tissue or an artificial lung matrix.
9. The method of claim 1, wherein the bioartificial lung comprises
a sufficient number of cells to provide full lung function or a
fraction thereof.
Description
TECHNICAL FIELD
This document provides an apparatus and methods related to tissue
generation. For example, this document provides methods for
generating transplantable lung tissue in a human or animal
subject.
BACKGROUND
Lung transplants represent a final hope for many patients
experiencing conditions typified by lung failure, e.g., Chronic
obstructive pulmonary disease (COPD)COPD, Cystic Fibrosis, lung
cancers, and congenital lung diseases, among others. Typical wait
time for a lung transplant can be two years or more, resulting in a
30% mortality rate for those on the waiting list.
SUMMARY
Presented is an airway organ bioreactor apparatus. The apparatus
has an organ chamber configured to hold an organ matrix scaffold
onto which a cell media is perfused to grow an organ. The apparatus
further has a wet ventilator system configured to supply a wet
ventilation to the organ via the first branch of the connector. The
apparatus further has a dry ventilator system configured to supply
a dry ventilation to the organ via the first branch of the
connector. The apparatus further has a controller configured to
control the delivery of wet ventilation or delivery of dry
ventilation.
The apparatus can further comprise a connector including a first
branch, a second branch, and a third branch connected to the organ.
The apparatus further has a first three-way junction at which the
first branch of the connector and the second branch of the
connector are connected with the third branch of the connector. The
three-way junction including a switch can be configured to toggle
between the first branch and the second branch. The apparatus
further has a wet ventilator system configured to supply a wet
ventilation to the organ via the first branch of the connector. The
apparatus further has a dry ventilator system configured to supply
a dry ventilation to the organ via the second branch of the
connector. The apparatus further has a controller configured to
control the switch of the first three-way junction, thereby
controlling delivery of wet ventilation or delivery of dry
ventilation.
The apparatus can further comprise a reservoir system configured to
supply cell media to the organ over an ingress line; and drain
waste media from the organ over an egress line, the egress line
including a first branch, a second branch, and a third branch; and
a second three-way junction at which the first branch of the egress
line and the second branch of the egress line are connected with
the third branch of the egress line. The wet ventilator system can
comprise a wet ventilator connected to the organ chamber via a wet
ventilation line; and a compliance chamber connected to the organ
via the first branch of the connector. A wet positive and
expiratory pressure (wPEEP) can be provided to the organ chamber
via an elevation of the compliance chamber. The apparatus can
further comprise an afterload chamber connected to the organ
chamber via the second branch of the egress line; and the reservoir
system via an egress return line. The reservoir system can comprise
a first reservoir connected to the organ chamber via an ingress
line; and a second reservoir connected to the organ chamber via an
organ chamber drain; and the afterload chamber via the egress
return line, wherein the first reservoir and second reservoir
circulate media over a reservoir feed line and a reservoir drain.
The dry ventilator system can comprise a dry ventilation chamber
including a nebulizer, connected to the organ via the second branch
of the connector; and a first dry ventilator configured to provide
a dry positive and expiratory pressure (dPEEP) to the organ chamber
and connected to the dry ventilation chamber via a dPEEP line. The
dry ventilator system can further comprise a second dry ventilator
connected to the organ chamber via a dry ventilator line. The
apparatus can further comprise a gas tank configured to supply
gaseous media to the organ chamber, the dry ventilation chamber,
and the reservoir system. The controller can be operated by a
computer.
In another aspect, this document features a method of providing a
bioartificial airway organ. The method can comprise providing a
lung tissue matrix comprising a lung tissue matrix and substantial
vasculature; seeding the lung tissue matrix with cells; providing
the organ with wet ventilation for a time sufficient for a first
desired degree of organ maturation to occur; and providing the
wet-matured organ with dry ventilation for a time sufficient for a
second desired degree of organ maturation to occur, thereby
providing a bioartificial lung. The method can further comprise
seeding the lung tissue matrix with endothelial cells over the
vasculature of the organ; and seeding the airway lung tissue matrix
with epithelial cells over an airway of the organ. The method can
further comprise seeding the lung tissue matrix with stem cells
over a vasculature of the organ. The stem cells can be bone marrow
derived mesenchymal stem cells or induced pluripotent stem (iPS)
cells. The stem cells can be suspended in a fluid at a
concentration of about 100 million cells per 30 cc of fluid. The
endothelial cells can be suspended in a fluid at a concentration of
about 100 million cells per 10 cc of fluid. The epithelial cells
can be suspended in a fluid at a concentration of about 100 million
cells per 5 cc of fluid. The method can further comprise monitoring
the degree of organ maturation until the first desired degree of
organ maturation has occurred; stopping the providing of the wet
ventilation to the organ; applying an artificial surfactant to the
organ; and starting the providing of the dry ventilation to the
organ. Providing the lung tissue matrix with wet ventilation can
comprise connecting the airway to a wet ventilator via a wet
ventilator line; connecting the organ to a compliance chamber via a
wet ventilation line; increasing a wet airway pressure over the wet
ventilation line; and providing a wet positive and expiratory
pressure (wPEEP) to the organ by elevating the compliance chamber.
The wet ventilation is provided at a physiologic tidal volume.
Providing the wet-matured organ with dry ventilation can comprise
connecting the airway to a dry ventilation chamber via a dry
ventilation line; connecting the dry ventilation chamber to a first
dry ventilator over a dry positive and expiratory pressure (dPEEP)
line; increasing a dry airway pressure over the dry ventilation
line; disconnecting the wet ventilation line; and connecting the
organ to a second dry ventilator via a dry ventilator line. The
lung tissue matrix can comprise decellularized human lung tissue or
an artificial lung matrix. The bioartificial lung can comprise a
sufficient number of cells to provide full lung function or a
fraction thereof.
In another aspect, this document features a bioartificial lung
produced by the method provided herein. The bioartificial lung can
be a full lung or a portion thereof.
In a further aspect, this document features a method of treating a
subject having impaired or reduced lung capacity. The method can
comprise transplanting the bioartificial lung into the subject.
Unless otherwise defined, all technical and scientific terms used
herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention pertains.
Although methods and materials similar or equivalent to those
described herein can be used to practice the invention, suitable
methods and materials are described below. All publications, patent
applications, patents, and other references mentioned herein are
incorporated by reference in their entirety. In case of conflict,
the present specification, including definitions, will control. In
addition, the materials, methods, and examples are illustrative
only and not intended to be limiting.
The details of one or more embodiments of the invention are set
forth in the accompanying drawings and the description below. Other
features, objects, and advantages of the invention will be apparent
from the description and drawings, and from the claims.
DESCRIPTION OF DRAWINGS
FIG. 1 is a schematic diagram of an exemplary lung bioreactor.
FIGS. 2A, 2B, 2C, and 2D are flow charts of an exemplary method for
growing lung tissue in a lung bioreactor.
FIG. 3 is a schematic drawing of an exemplary lung
decellularization unit.
FIG. 4 is a schematic drawing of an exemplary lung bioreactor in
cell seeding mode.
FIG. 5 is a schematic drawing of an exemplary lung bioreactor in
perfusion mode.
FIG. 6 is a schematic drawing of an exemplary lung bioreactor.
DETAILED DESCRIPTION
This document relates to methods and materials involved in organ
generation. The present invention is based, at least in part, on
the discovery of bioreactors configured to generate functional lung
tissue that can be used to provide a more realistic environment for
growth of functional airway organs ready for transplantation into
humans and other animals. The lung tissue is generated over a given
matrix, e.g., an artificial or decellularized lung tissue
matrix.
As used herein, a "functional" lung tissue performs most or all of
the functions of a normal healthy lung, e.g., allows for
transportation of oxygen from the air into the bloodstream, and the
release of carbon dioxide from the bloodstream into the air. It
humidifies the inhaled air, produces surfactant to decrease surface
tension in the alveoli and produces and transports mucus to remove
inhaled particulate matter from the distal to the proximal
airway.
As used herein, the terms "decellularized" and "acellular" are used
interchangeably and are defined as the complete or near complete
absence of detectable intracellular, endothelial cells, epithelial
cells, and nuclei in histologic sections using standard
histological staining procedures. Preferably, but not necessarily,
residual cell debris also has been removed from the decellularized
organ or tissue.
Decellularized Tissue/Organ Matrices
Methods and materials for a preparing a decellularized lung tissue
matrix are known in the art. Any appropriate materials can be used
to prepare such a matrix. In a preferred embodiment, a tissue
matrix can be an acellular tissue scaffold developed from
decellularized lung tissue. For example, tissue such as human
lungs, or a portion thereof, can be decellularized by an
appropriate method to remove native cells from the tissue while
maintaining morphological integrity and vasculature of the tissue
or tissue portion and preserving extracellular matrix (ECM)
proteins. In some cases, cadaveric lungs, or portions thereof, can
be used. Decellularization methods can include subjecting tissue
(e.g., lung tissue) to repeated freeze-thaw cycles using liquid
nitrogen. In other cases, a tissue can be subjected to an anionic
or ionic cellular disruption medium such as sodium dodecyl sulfate
(SDS), polyethylene glycol (PEG), or TritonX-100. The tissue can
also be treated with a nuclease solution (e.g., ribonuclease,
deoxyribonuclease) and washed in sterile phosphate buffered saline
with mild agitation. In some cases, decellularization can be
performed by cannulating the vessels, ducts, and/or cavities of the
organ or tissue using methods and materials known in the art.
Following the cannulating step, the organ or tissue can be perfused
via the cannula with a cellular disruption medium as described
above. Perfusion through the tissue can be antegrade or retrograde,
and directionality can be alternated to improve perfusion
efficiency. Depending upon the size and weight of an organ or
tissue and the particular anionic or ionic detergent(s) and
concentration of anionic or ionic detergent(s) in the cellular
disruption medium, a tissue generally is perfused from about 2 to
about 12 hours per gram of tissue with cellular disruption medium.
Including washes, an organ may be perfused for up to about 12 to
about 72 hours per gram of tissue. Perfusion generally is adjusted
to physiologic conditions including flow rate and pressure.
Decellularized tissue can consist essentially of the extracellular
matrix (ECM) component of all or most regions of the tissue,
including ECM components of the vascular tree. ECM components can
include any or all of the following: fibronectin, fibrillin,
laminin, elastin, members of the collagen family (e.g., collagen I,
III, and IV), glycosaminoglycans, ground substance, reticular
fibers and thrombospondin, which can remain organized as defined
structures such as the basal lamina. In a preferred embodiment,
decellularized lung tissue matrix retains a substantially intact
vasculature. Preserving a substantially intact vasculature enables
connection of the tissue matrix to a subject's vascular system upon
transplantation. In addition, a decellularized tissue matrix can be
further treated with, for example, irradiation (e.g., UV, gamma) to
reduce or eliminate the presence of any type of microorganism
remaining on or in a decellularized tissue matrix.
Methods for obtaining decellularized tissue matrices using
physical, chemical, and enzymatic means are known in the art, see,
e.g., Liao et al, Biomaterials 29(8):1065-74 (2008); Gilbert et
al., Biomaterials 27(9):3675-83 (2006); Teebken et al., Eur. J.
Vasc. Endovasc. Surg. 19:381-86 (2000). See also U.S. Pat.
Publication Nos. 2009/0142836; 2005/0256588; 2007/0244568; and
2003/0087428.
Artificial Organ Matrices
Methods and materials for a preparing an artificial organ matrix
are known in the art. Any appropriate materials can be used to
prepare such a matrix. In a preferred embodiment, an artificial
organ matrix can be a scaffold developed from porous materials such
as, for example, polyglycolic acid, Pluronic F-127 (PF-127),
Gelfoam sponge, collagen-glycosaminoglycan (GAG),
fibrinogen-fibronectin-vitronectin hydrogel (FFVH), and elastin.
See, e.g., Ingenito et al., J Tissue Eng Regen Med. 2009 Dec. 17;
Hoganson et al., Pediatric Research, May 2008, 63(5):520-526; Chen
et al., Tissue Eng. 2005 September-October; 11(9-10):1436-48. In
some cases, an artificial organ matrix can have porous structures
similar to alveolar units. See Andrade et al., Am J Physiol Lung
Cell Mol. Physiol. 2007 February; 292(2):L510-8. In some cases, an
implanted artificial organ matrix can express organ-specific
markers (e.g., lung-specific markers for Clara cells, pneumocytes,
and respiratory epithelium). In some cases, an implanted artificial
organ matrix can organize into identifiable structures (e.g.,
structures similar to alveoli and terminal bronchi in an artificial
lung matrix). For example, an implanted artificial lung maxtrix
made using FFVH can promote cell attachment, spreading and
extracellular matrix expression in vitro and apparent engraftment
in vivo, with evidence of trophic effects on the surrounding
tissue. See Ingenito et al., supra. See also U.S. Pat. Nos.
7,662,409 and 6,087,552; United States Patent Publication Nos.
2010/0034791; 2009/0075282; 2009/0035855; 2008/0292677;
2008/0131473; 2007/0059293; 2005/0196423; 2003/0166274;
2003/0129751; 2002/0182261; 2002/0182241; and 2002/0172705.
Cell Seeding
In the methods described herein, a lung tissue matrix, e.g.,
decellularized lung tissue matrix or artificial lung matrix, is
seeded with cells, e.g., differentiated or regenerative cells.
Any appropriate regenerative cell type, such as naive or
undifferentiated cell types, can be used to seed the lung tissue
matrix. As used herein, regenerative cells can include, without
limitation, progenitor cells, precursor cells, and "adult"-derived
stem cells including umbilical cord cells (e.g., human umbilical
vein endothelial cells) and fetal stem cells. Regenerative cells
also can include differentiated or committed cell types. Stem cells
appropriate for the methods and materials provided herein can
include human induced pluripotent stem cells (iPSC), mesenchymal
stem cells, human umbilical vein endothelial cells, multipotent
adult progenitor cells (MAPC), or embryonic stem cells. In some
cases, regenerative cells derived from other tissues also can be
used. For example, regenerative cells derived from skin, bone,
muscle, bone marrow, synovium, or adipose tissue can be used to
develop stem cell-seeded tissue matrices.
In some cases, a lung tissue matrix provided herein can be further
seeded with differentiated cell types such as human epithelial
cells and endothelial cells. For example, a lung matrix can be
seeded with endothelial cells via the vasculature, and epithelial
and mesenchymal cells, and human umbilical vein endothelial cells
(HUVEC) through perfusion seeding.
Any appropriate method for isolating and collecting cells for
seeding can be used. For example, induced pluripotent stem cells
generally can be obtained from somatic cells "reprogrammed" to a
pluripotent state by the ectopic expression of transcription
factors such as Oct4, Sox2, Klf4, c-MYC, Nanog, and Lin28. See
Takahashi et al., Cell 131:861-72 (2007); Park et al., Nature
451:141-146 (2008); Yu et al., Science 318:1917-20 (2007). Cord
blood stem cells can be isolated from fresh or frozen umbilical
cord blood. Mesenchymal stem cells can be isolated from, for
example, raw unpurified bone marrow or ficoll-purified bone marrow.
Epithelial and endothelial cells can be isolated and collected from
living or cadaveric donors, e.g., from the subject who will be
receiving the bioartificial lung, according to methods known in the
art. For example, epithelial cells can be obtained from a skin
tissue sample, and endothelial cells can be obtained from a
vascular tissue sample. In some embodiments, proteolytic enzymes
are perfused in to the tissue sample through a catheter placed in
the vasculature. Portions of the enzymatically treated tissue can
be subjected to further enzymatic and mechanical disruption. The
mixture of cells obtained in this manner can be separated to purify
epithelial and endothelial cells. In some cases, flow
cytometry-based methods (e.g., fluorescence-activated cell sorting)
can be used to sort cells based on the presence or absence of
specific cell surface markers. In cases where non-autologous cells
are used, the selection of immune type-matched cells should be
considered, so that the organ or tissue will not be rejected when
implanted into a subject.
Isolated cells can be rinsed in a buffered solution (e.g.,
phosphate buffered saline) and resuspended in a cell culture
medium. Standard cell culture methods can be used to culture and
expand the population of cells. Once obtained, the cells can be
contacted with a tissue matrix to seed the matrix. For example, a
tissue matrix can be seeded with at least one cell type in vitro at
any appropriate cell density. For example, cell densities for
seeding a matrix can be at least 1.times.10.sup.3 cells/gram
matrix. Cell densities can range between about 1.times.10.sup.5 to
about 1.times.10.sup.10 cells/gram matrix (e.g., at least 100,000,
1,000,000, 10,000,000, 100,000,000, 1,000,000,000, or
10,000,000,000 cells/gram matrix) can be used.
In some cases, a decellularized or artificial lung tissue matrix as
provided herein can be seeded with the cell types and cell
densities described above by perfusion seeding. For example, a flow
perfusion system can be used to seed the decellularized lung tissue
matrix via the vascular system preserved in the tissue matrix. In
some cases, automated flow perfusion systems can be used under the
appropriate conditions. Such perfusion seeding methods can improve
seeding efficiencies and provide more uniform distribution of cells
throughout the composition. Quantitative biochemical and image
analysis techniques can be used to assess the distribution of
seeded cells following either static or perfusion seeding
methods.
In some cases, a tissue matrix can be impregnated with one or more
growth factors to stimulate differentiation of the seeded
regenerative cells. For example, a tissue matrix can be impregnated
with growth factors appropriate for the methods and materials
provided herein, for example, vascular endothelial growth factor
(VEGF), TGF-.beta. growth factors, bone morphogenetic proteins
(e.g., BMP-1, BMP-4), platelet derived growth factor (PDGF), basic
fibroblast growth factor (b-FGF), e.g., FGF-10, insulin-like growth
factor (IGF), epidermal growth factor (EGF), or growth
differentiation factor-5 (GDF-5). See, e.g., Desai and Cardoso,
Respir. Res. 3:2 (2002).
Seeded tissue matrices can be incubated for a period of time (e.g.,
from several hours to about 14 days or more) post-seeding to
improve fixation and penetration of the cells in the tissue matrix.
The seeded tissue matrix can be maintained under conditions in
which at least some of the regenerative cells can multiply and/or
differentiate within and on the acellular tissue matrix. Such
conditions can include, without limitation, the appropriate
temperature and/or pressure, electrical and/or mechanical activity
(e.g., ventilation), force, the appropriate amounts of O.sub.2
and/or CO.sub.2, an appropriate amount of humidity, and sterile or
near-sterile conditions. Such conditions can also include wet
ventilation, wet to dry ventilation and dry ventilation. In some
cases, nutritional supplements (e.g., nutrients and/or a carbon
source such as glucose), exogenous hormones, or growth factors can
be added to the seeded tissue matrix. Histology and cell staining
can be performed to assay for seeded cell propagation. Any
appropriate method can be performed to assay for seeded cell
differentiation. In general, the methods described herein will be
performed in an airway organ bioreactor apparatus, e.g., as
described herein.
Thus the methods described herein can be used to generate a
transplantable bioartificial lung tissue, e.g., for transplanting
into a human subject. As described herein, a transplantable tissue
will preferably retain a sufficiently intact vasculature that can
be connected to the patient's vascular system.
The bioartificial lung tissues described herein can be combined
with packaging material to generate articles of manufacture or
kits. Components and methods for producing articles of manufacture
are well known. In addition to the bioartificial tissues, an
article of manufacture or kit can further can include, for example,
one or more anti-adhesives, sterile water, pharmaceutical carriers,
buffers, and/or other reagents for promoting the development of
functional lung tissue in vitro and/or following transplantation.
In addition, printed instructions describing how the composition
contained therein can be used can be included in such articles of
manufacture. The components in an article of manufacture or kit can
be packaged in a variety of suitable containers.
Methods for Using Bioartificial Lungs
This document also provides methods and materials for using
bioartificial lung tissues and, in some cases, promoting lung
function. In some embodiments, the methods provided herein can be
used to restore some lung function in patients having diseases that
impair or reduce lung capacity (e.g., cystic fibrosis, COPD,
emphysema, lung cancer, asthma, lung trauma, or other genetic or
congenital lung abnormalities, e.g., bronchogenic cyst, pulmonary
agenesis and hypoplasia, polyalveolar lobe, alveolocapillary
dysplasia, sequestration including arteriovenous malformation (AVM)
and scimitar syndrome, pulmonary lymphangiectasis, congenital lobar
emphysema (CLE), and cystic adenomatoid malformation (CAM) and
other lung cysts). The methods provided herein also include those
wherein the subject is identified as in need of a particular stated
treatment, e.g., increased lung function, or increased or improved
lung capacity.
Bioartificial lung tissues (e.g., whole organs or portions thereof)
can be generated according to the methods provided herein. In some
embodiments, the methods comprise transplanting a bioartificial
lung tissue as provided herein to a subject (e.g., a human patient)
in need thereof. In some embodiments, a bioartificial lung tissue
is transplanted to the site of diseased or damage tissue. For
example, bioartificial lung tissues can be transplanted into the
chest cavity of a subject in place of (or in conjunction with) a
non-functioning or poorly-functioning lung; methods for performing
lung transplantation are known in the art, see, e.g., Boasquevisque
et al., Surgical Techniques: Lung Transplant and Lung Volume
Reduction, Proceedings of the American Thoracic Society 6:66-78
(2009); Camargo et al., Surgical maneuvers for the management of
bronchial complications in lung transplantation, Eur J Cardiothorac
Surg 2008; 34:1206-1209 (2008); Yoshida et al., "Surgical Technique
of Experimental Lung Transplantation in Rabbits," Ann Thorac
Cardiovasc Surg. 11(1):7-11 (2005); Venuta et al., Evolving
Techniques and Perspectives in Lung Transplantation,
Transplantation Proceedings 37(6):2682-2683 (2005); Yang and Conte,
Transplantation Proceedings 32(7):1521-1522 (2000); Gaissert and
Patterson, Surgical Techniques of Single and Bilateral Lung
Transplantation in The Transplantation and Replacement of Thoracic
Organs, 2d ed. Springer Netherlands (1996).
The methods can include transplanting a bioartificial lung or
portion thereof as provided herein during a surgical procedure to
partially or completely remove a subject's lung and/or during a
lung resection. In some cases, the methods provided herein can be
used to replace or supplement lung tissue and function in a
subject, e.g., a human or animal subject.
Any appropriate method(s) can be performed to assay for lung
function before or after transplantation. For example, methods can
be performed to assess tissue healing, to assess functionality, and
to assess cellular in-growth. In some cases, tissue portions can be
collected and treated with a fixative such as, for example, neutral
buffered formalin. Such tissue portions can be dehydrated, embedded
in paraffin, and sectioned with a microtome for histological
analysis. Sections can be stained with hematoxylin and eosin
(H&E) and then mounted on glass slides for microscopic
evaluation of morphology and cellularity. For example, histology
and cell staining can be performed to detect seeded cell
propagation. Assays can include functional evaluation of the
transplanted tissue matrix or imaging techniques (e.g., computed
tomography (CT), ultrasound, or magnetic resonance imaging (e.g.,
contrast-enhanced MRI)). Assays can further include functional
tests under rest and physiologic stress (e.g., body pletysmography,
lung function testing). Functionality of the matrix seeded with
cells can be assayed using methods known in the art, e.g.,
histology, electron microscopy, and mechanical testing (e.g., of
volume and compliance). Gas exchange can be measured as another
functionality assay. To assay for cell proliferation, thymidine
kinase activity can be measured by, for example, detecting
thymidine incorporation. In some cases, blood tests can be
performed to evaluate the function of the lungs based on levels of
oxygen in the blood.
In some cases, molecular biology techniques such as RT-PCR can be
used to quantify the expression of metabolic and differentiation
markers. Any appropriate RT-PCR protocol can be used. Briefly,
total RNA can be collected by homogenizing a biological sample
(e.g., tendon sample), performing a chloroform extraction, and
extracting total RNA using a spin column (e.g., RNeasy.RTM. Mini
spin column (QIAGEN, Valencia, Calif.)) or other nucleic
acid-binding substrate. In other cases, markers associated with
lung cells types and different stages of differentiation for such
cell types can be detected using antibodies and standard
immunoassays.
Airway Organ Bioreactor Apparatus
An exemplary airway organ bioreactor apparatus is presented in FIG.
1. Throughout the specification, a lung will be offered as an
example of an airway organ. Other examples can include, e.g., a
trachea.
Referring to FIG. 1, components of the bioreactor include a lung
chamber 102, an airway connector including a tracheal line 124, a
wet ventilation line 150, and a dry ventilation line 134, wet
ventilator system 120, dry ventilator system 116 and 118, three-way
connector 148 at the junction between the tracheal line 124, wet
ventilation line 150, and dry ventilation line 134, and controller
(not shown). The controller is computer-operated, but also be
operated manually. The bioreactor can also include a pulmonary
arterial line 122, a pulmonary venous line 126, a reservoir system
104 and 106, a roller pump 114, a gas tank 122 and accompanying gas
lines, an afterload chamber 110, a pulmonary venous return line
136, and a lung chamber pressure line 128. The bioreactor further
includes a compliance chamber 109 and compliance chamber drain 146.
The bioreactor can further include a membrane oxygenator in
addition or instead to provide oxygenation and carbonation of
perfusing media solutions (not shown).
Lung chamber 102 holds a decellularized lung matrix scaffold. Lung
chamber 102 is closed to provide a sterile lung culture
environment. The pulmonary artery of the lung matrix is connected
to pulmonary arterial line 122 and the pulmonary vein of the lung
matrix is connected to pulmonary arterial vein 126, each via
vascular cannulas. The trachea of the lung matrix is connected to
the airway connector via the tracheal line 124.
Within lung chamber 102, the cell matrix is perfused antegradely
with a cell media in order to allow seeding of cells to grow the
lung. The perfusion takes place over pulmonary arterial line 122 to
the pulmonary artery. From there, media flows through the pulmonary
vasculature and flows out to the reservoir system (104 and
106).
Reservoir system includes a first reservoir 104 and a second
reservoir 106, as well as a reservoir feeding line 140 and a
reservoir drain (140). Cell media circulates between the reservoirs
104 and 106 through reservoir feeding line 138 and reservoir drain
140. A microfilter can optionally be placed in feeding line 140 for
sterile filtration. Cell media is also oxygenated in reservoirs 104
and 106. For perfusion, the cell media is fed from reservoir 104
through the pulmonary arterial line 122 via roller pump 114 or via
gravity to the pulmonary artery.
The media that flows out to reservoir 106 is aspirated directly
from lung chamber 102 via lung chamber drain (4) to maintain a
constant fluid level within the lung chamber 102. The media that
flows out of the lung via the third connector 126 drains to the
afterload chamber 110 via gravity and is aspirated via the
afterload chamber drain 136 to the reservoir 106. Afterload chamber
110 is connected to the lung chamber 102 via the lung chamber
pressure line 130 and the reservoir 106 via pulmonary venous return
line 136. The lung chamber pressure line equilibrates the pressures
in lung chamber 102 and afterload chamber 110. Afterload chamber
110 is also connected to the lung chamber 102 via the trachea and
wet ventilation line through three-way junction 156.
One exemplary method of reintroducing cells into the matrix is as
follows. During a perfusion of the lung matrix, a cellularization
of the matrix begins. About 100 million mesenchymal cells suspended
in about 30 cc of media are seeded via pulmonary arterial line 122.
The mesenchymal cells are bone marrow derived embryo stem cells,
but can also be e.g., iPS or hematopoietic cells as described in
U.S. Ser. No. 12/233,017, "Generation of Inner Ear Cells," the
contents of which are incorporated in their entirety by reference.
In some embodiments, upon completion of the seeding, the perfusion
is stopped, e.g., for about 60 minutes, to allow for cell
attachment. During the stoppage of the perfusion, cell media is
drained from the trachea and pulmonary vein; the drained cell media
then flows to reservoir 106. After the 60 minute stoppage, the
perfusion with media alone is continued, e.g., for about 24 hours.
To maintain constant media level in compliance chamber 109, it can
be connected to reservoir 104 via an additional line (not
shown).
Next, conditions are set up for seeding of endothelial cells. The
tracheal line 124 is connected to the wet ventilation line 150 via
the three-way junction 148 and its controller. The three-way
junction 156 is turned, using its controller, to connect the wet
ventilation line 150 to the compliance chamber 109. Compliance
chamber 109 provides positive wet airway pressure (wAP) to limit
net media flow through interstitial space and the trachea while
limiting airway pressure to a physiologic range. The wAP is
adjusted through an adjustment of the chamber 109. As a result, a
portion of the cell media drains through the pulmonary venous line
(3) to reservoir 106, while a smaller portion of the cell media
drains via lymphatics through lung chamber drain line 128 to
reservoir 106.
About 100 million endothelial cells suspended in about 15 cc of
media are seeded through pulmonary arterial line 122 via about 10
minutes of gravity feeding. Upon completion of the seeding, the
perfusion is stopped, e.g., for about 60 minutes to allow for cell
attachment. After the stoppage, the perfusion is continued, e.g.,
for about 3-5 days to allow for a formation of an endothelial cell
monolayer.
After endothelial cells have been seeded and the endothelial cell
monolayer has been formed, epithelial cells are ready to be seeded.
For seeding of epithelial cells, the three-way junction 156 is
turned, using its controller, to occlude wet ventilation line 150.
About 200 million epithelial cells suspended in about 15 cc of
media are seeded through tracheal line 124 into the trachea. In
some embodiments, the epithelial cells are pneumocytes. Upon
completion of the seeding, perfusion via the pulmonary artery is
stopped, e.g., for about 60 minutes.
Also, once cell seeding of the lung has been completed, wet
ventilation is needed to advance cell suspension into the
peripheral airways. Wet ventilator system 120 provides wet
ventilation to the lung over wet ventilation line 150.
Three-way junction 156 is turned, using its controller, to connect
wet ventilation line 150 to compliance chamber 109. The wAP is
increased to provide a small flow into the interstitial space and
increase cell attachment. Wet ventilation is provided to the lung
for about 5 minutes, held for about 60 minutes, then provided to
the lung for about another 5 minutes, and then held for about 24
hours. The wet ventilation is provided at physiologic tidal volume
(about 500 mL for a human) while at a reduced rate to keep wet peak
inspiratory and expiratory pressure low. A wet positive and
expiratory pressure is provided via elevation of compliance chamber
109.
Once perfusion is resumed, antegrade perfusion and wet ventilation
are provided for a period of about 5 days to enable tissue
formation.
A switch from wet to dry ventilation is made after the about 5 day
period or after a monitor (not shown) determines that the lung has
reached sufficient maturity. Artificial surfactant is administered
via tracheal line 124. Then three-way junction 148 is turned, using
its controller, so that the tracheal line 124 is connected to dry
ventilation line 150 and dry ventilation system (116 and 118). Dry
ventilation system includes dry ventilation chamber 112 having a
nebulizer (not shown) for providing humidified air, first dry
ventilator 116, and second dry ventilator 118. Dry ventilation
chamber 112 is connected to first dry ventilator 116 via a dry PEEP
line 144 and to the tracheal line via the dry ventilation line 150.
This way, the lung is ventilated to slowly fill its airspace with
gas rather than fluid. The gas used is carbogen supplied via gas
line by gas tank 122. Dry ventilator 116 is configured to provide a
dPEEP to dry ventilation chamber 112 and subsequently enable fluid
drainage in lung chamber 102.
Next, wet ventilation system 120 is discontinued and dry ventilator
118 is opened to the lung chamber in order to increase ventilation
rate to the physiologic rate, empty lung chamber 102 of fluid, and
surround lung with humidified air within lung chamber 102. After
about 3 days of tissue maturation, a perfusate gas analysis is
performed to confirm formation of functional tissue and that the
lung can be removed from the bioreactor.
In switching between the wet ventilation and dry ventilation, the
lung develops under conditions simulating the conditions under
which a lung develops naturally. It has been determined that this
environment is necessary for lung development, and that the
bioreactor as described provides system and methods needed to
generate tissue engineered lungs for transplantation.
An exemplary method of cellularizing a lung matrix is illustrated
in FIG. 2A. A lung matrix is placed 210 into the lung chamber. The
lung matrix is then perfused 220 with a cell media over a pulmonary
arterial line. The lung matrix is then provided 230 with wet
ventilation. Finally, the lung matrix is provided with dry
ventilation 240.
During perfusion 220 of the lung matrix, as illustrated in FIG. 2B,
the lung matrix is seeded 222 with mesenchymal or other stem cells
over the vasculature of the lung. The lung matrix is then seeded
224 with endothelial cells over the vasculature of the lung. The
lung matrix is then seeded 226 with epithelial cells over the lung
airway.
To provide the wet ventilation, as illustrated in FIG. 2C, the lung
airway is connected 231 to a wet ventilator over a wet ventilator
line. The lung is then connected 232 to a compliance chamber via a
wet ventilation line. By adjusting the height of a fluid within the
compliance chamber, a wet airway pressure is then increased 233
over the wet ventilation line. Further, by elevating the compliance
chamber, a wet PEEP (wPEEP) is then provided 234. A degree of
maturation of the lung growing on the lung matrix is monitored 235.
If the degree of maturation is determined 236 to be acceptable,
then a transition to a dry ventilation is begun 237.
To provide the dry ventilation, as illustrated in FIG. 2D, an
artificial surfactant is applied 241 through the trachea. The
trachea is then connected 242 to a dry ventilation chamber via a
dry ventilation line. The dry ventilation chamber is connected 243
to a first dry ventilator over a dPEEP line. A dry airway pressure
is then increased 244 over the dry ventilation line. The wet
ventilation line is 245 disconnected, and the lung chamber is
connected 246 to a second dry ventilator.
A complete rat lung may require, in total, between about 200 and
about 400 million cells. Extrapolation to human provides an
estimate of between about 20 and about 40 billion cells for a
complete lung. Such a number of cells to generate may require more
time than a patient may have. A patient that requires a percentage
of lung function, say, 20%, would only need about 20% of this
number and would have to wait proportionally less time for a new
lung.
An exemplary airway organ bioreactor apparatus for use in organ
decellularization is presented in FIG. 3. As described above, a
lung will be offered as an example of an airway organ. Referring to
FIG. 3, components of the bioreactor include lung chamber 302,
sealed gravity reservoir 304, and large reservoir 306, where
reservoirs 304 and 306 contain decellularization solution for
perfusion into the lung in lung chamber 302. Decellularization
solution circulates between the reservoirs 304 and 306 through a
reservoir feeding line 314. The pulmonary artery of the lung matrix
is connected to pulmonary arterial line 308 through which
decellularization solution is perfused into lung tissue via gravity
flow from reservoir 304. Following decellularization, waste is
removed from lung chamber 302. In some cases, solution from lung
chamber 302 is recirculated via pump 312 which feeds into reservoir
306.
An exemplary airway organ bioreactor apparatus for use in cell
seeding is presented in FIG. 4. Referring to FIG. 4, components of
the bioreactor include lung chamber 402, sealed gravity reservoir
404, and large reservoir 406, where reservoirs 404 and 406 contain
cell media for perfusion into the lung in lung chamber 402. Cell
media circulates between reservoirs 404 and 406 through reservoir
feeding line 408. The pulmonary artery of the lung matrix is
connected to pulmonary arterial line 410 and the pulmonary vein of
the lung matrix is connected to pulmonary vein line 412, each via
vascular cannulas. For cell seeding, cell media is fed from
reservoir 404 through the pulmonary arterial line 410 via a pump or
via gravity to the pulmonary artery. Media that flows out of the
lung via the third connector 412 (venous outflow) drains to
afterload compliance chamber 420 via gravity and is aspirated via
afterload chamber drain to reservoir 406. Afterload chamber 420 is
connected to the lung chamber 402 via the lung chamber pressure
line and the reservoir 406 via pulmonary venous return line 412.
The lung chamber pressure line equilibrates the pressures in lung
chamber 402 and afterload chamber 420. Afterload chamber 420 is
also connected to the lung chamber 402 via the trachea and wet
ventilation line through three-way junction 414.
The tracheal line is connected to wet ventilation line via
three-way junction 414 and its controller. Three-way junction 414
is turned, using its controller, to connect the wet ventilation
line to the compliance chamber 420. Compliance chamber 420 provides
positive wet airway pressure (wAP) to limit net media flow through
interstitial space and the trachea while limiting airway pressure
to a physiologic range. The wAP is adjusted through an adjustment
of the chamber 420. As a result, a portion of the cell media drains
through the pulmonary venous line 412 to reservoir 406, while a
smaller portion of the cell media drains via lymphatics through the
lung chamber drain line to reservoir 406.
An exemplary airway organ bioreactor apparatus for use in matrix
perfusion is presented in FIG. 5. Referring to FIG. 5, components
of the bioreactor include lung chamber 502, sealed gravity
reservoir 504, and large reservoir 506, where reservoirs 504 and
506 contain perfusion solution (e.g., blood) for perfusion into the
lung in lung chamber 502. Cell media circulates between the
reservoirs 504 and 506 through reservoir feeding line 508. The
pulmonary artery of the lung matrix is connected to pulmonary
arterial line 510 and the pulmonary vein of the lung matrix is
connected to pulmonary arterial vein 512, each via vascular
cannulas. Following cell seeding, wet ventilation is needed to
advance cell suspension into the peripheral airways. Wet ventilator
system 518 provides wet ventilation to the lung over wet
ventilation line. Then three-way junction 514 is turned, using its
controller, so that the tracheal line is connected to dry
ventilation line and dry ventilation system 516.
An exemplary airway organ bioreactor apparatus is presented in FIG.
6. Referring to FIG. 6, components of the bioreactor include lung
chamber 602, sealed gravity reservoir 616, and large reservoir 606,
where reservoirs 616 and 606 contain perfusion solution for
perfusion into the lung in lung chamber 602. Solution circulates
between the reservoirs 616 and 606 through a reservoir feeding
line. The pulmonary artery of the lung matrix is connected to
pulmonary arterial line 626 and the pulmonary vein of the lung
matrix is connected to pulmonary arterial vein 628, each via
vascular cannulas. For cell seeding, media is fed from reservoir
616 through the pulmonary arterial line 626 via a pump or via
gravity to the pulmonary artery. Media that flows out of the lung
via the third connector 628 (venous outflow) drains to afterload
compliance chamber 604 via gravity and is aspirated via afterload
chamber drain to reservoir 606. To maintain constant media level in
compliance chamber 604, it can be connected to reservoir 606 via an
additional line (not shown). Wet ventilator system 624 provides wet
ventilation to the lung over a wet ventilation line. Three-way
junction 620 is turned, using its controller, to connect wet
ventilation line to compliance chamber 604.
A switch from wet to dry ventilation is made after the about 5 day
period or after a monitor (not shown) determines that the lung has
reached sufficient maturity. Artificial surfactant is administered
via tracheal line 630. Then three-way junction 620 is turned, using
its controller, so that the tracheal line 630 is connected to dry
ventilation line and dry ventilation system 622. The lung is
ventilated to slowly fill its airspace with gas rather than
fluid.
The invention will be further described in the following examples,
which do not limit the scope of the invention described in the
claims.
EXAMPLE
Lung Regeneration Based on Perfusion Decellularized Matrix
Scaffolds
Lungs were isolated from heparinized adult SD rats (n=20) and
decellularized using detergent perfusion. Resulting extracellular
matrix (ECM) scaffolds were analyzed using histology, electron
microscopy and mechanical testing. Scaffolds were mounted in a
bioreactor and seeded with human umbilical cord endothelial cells
(HUVEC, n=4), HUVECs and human alveolar basal epithelial cells
(H-A549, n=4), and HUVECs and rat fetal lung cells (H-FLC, n=2).
Culture was maintained up to seven days. Lung function was analyzed
in an isolated lung apparatus using blood perfusion and
ventilation, normal lungs served as controls (n=4).
Perfusion decellularization of cadaveric lungs yielded acellular
lung ECM scaffolds with intact airway and vascular architecture.
Lung scaffolds could be repopulated with endothelial and epithelial
cells and maintained in a bioreactor. Gas exchange
(PaO.sub.2/FiO.sub.2 ratio) was lower in H-A549 constructs (103.6
mmHg), and equal in H-FLC constructs (455.1 mmHg) compared to
normal lung (465.8 mmHg). Compliance was reduced in decellularized
lungs (0.27 ml/cmH.sub.2O/s), but equal in H-FLC constructs (0.67
ml/cmH.sub.2O/s) and normal lung (0.69 ml/cmH.sub.2O/s).
Perfusion decellularization of cadaveric lungs yields intact whole
lung ECM scaffolds that can be seeded with epithelial and
endothelial cells to form bioartificial lungs with ventilation,
perfusion and gas exchange comparable to normal lungs.
Other Embodiments
It is to be understood that while the invention has been described
in conjunction with the detailed description thereof, the foregoing
description is intended to illustrate and not limit the scope of
the invention, which is defined by the scope of the appended
claims. Other aspects, advantages, and modifications are within the
scope of the following claims.
* * * * *